Poster at the 40th annual meeting of the Biopysical Society . Abstract: Biophys. J. 70, A211 (1996). Calcium cages chemically react after absorption of a UV photon to drastically increase the rate constant for release of Ca2+ (koff) from the cage. koff for photolytically modified cages can be >105 for some species (ref. 4), thus providing a means for controlling cellular chemistry with a temporal resolution sufficient to effect a wide range of cellular events. Calcium uncaging with a focused UV laser beam provides a diffraction-limited spot as narrow as 0.1 mm2 at the focal point; unfortunately, the axial resolution of UV excitation is relatively poor, resulting in calcium release throughout thick regions within cells. Consequently, the spatial resolution for calcium uncaging often is inadequate to probe the properies of cellular microdomains. Multi-photon excitation is intrinsically confined to a small region near (~1 ~m) the focal spot of the exciting laser beam. Consequently, calcium released from cages photolyzed with two-photon excitation will be initially confined to a three dimensionally defined volume (~1 femtoliter) at the focal spot. We have demonstrated the two-photon excited photorelease of calcium from three cages, NP-EGTA, DM-Nitrophen, and Azid-1, and quantified their two-photon action cross sections at a variety of wavelengths from 700 to 800 nm.